Integrins ��2��1 and ��11��1 regulate the survival of mesenchymal stem cells on collagen I

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors – α1β1, α2β1 and α11β1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2β1- or α11β1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.     

Lysine 63-linked Polyubiquitination of TAK1 at Lysine 158 Is Required for Tumor Necrosis Factor ��- and Interleukin-1��-induced IKK/NF-��B and JNK/AP-1 Activation

Transforming growth factor-β-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor α (TNFα)- and interleukin-1β (IL-1β)-induced IκB kinase (IKK)/nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) activation. Here we report that TNFα and IL-1β induce Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue within the kinase domain. Tumor necrosis factor receptor-associated factors 2 and 6 (TRAF2 and -6) act as the ubiquitin E3 ligases to mediate Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue in vivo and in vitro. Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue is required for TAK1-mediated IKK complex recruitment. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild type or a TAK1 mutant containing a K158R mutation revealed the importance of this site in TNFα and IL-1β-mediated IKK/NF-κB and JNK/AP-1 activation as well as IL-6 gene expression. Our findings demonstrate that Lys⁶³-linked polyubiquitination of TAK1 at Lys¹⁵⁸ is essential for its own kinase activation and its ability to mediate its downstream signal transduction pathways in response to TNFα and IL-1β stimulation.     

GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a ��-Arrestin 1-mediated ERK1/2 Activation in Pancreatic ��-Cells

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gα_s/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacological inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.     

Presynaptic Targeting of ��4��2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1��

The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders.The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs)⁵ regulate the release of neurotransmitters at presynaptic sites (1) and mediate fast synaptic transmission at postsynaptic sites of neurons (2). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4, 5).The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7, 8). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9) and show attenuated self-administration of nicotine (10). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive.Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15–17). The neurexins are encoded by three genes corresponding to neurexins I–III (18, 19), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20), to active zones of presynaptic terminals (21, 22). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABA_A receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24, 25). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26, 27). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.     

Targeting of Integrin ��1 and Kinesin 2�� by MicroRNA 183

MicroRNA 183 (miR-183) has been reported to inhibit tumor invasiveness and is believed to be involved in the development and function of ciliated neurosensory organs. We have recently found that expression of miR-183 increased after the induction of cellular senescence by exposure to H₂O₂. To gain insight into the biological roles of miR-183 we investigated two potential novel targets: integrin β1 (ITGB1) and kinesin 2α (KIF2A). miR-183 significantly decreased the expression of ITGB1 and KIF2A measured by Western blot. Targeting of the 3′-untranslated region (3′-UTR) of ITGB1 and KIF2A by miR-183 was confirmed by luciferase assay. Transfection with miR-183 led to a significant decrease in cell invasion and migration capacities of HeLa cells that could be rescued by expression of ITGB1 lacking the 3′-UTR. Although miR-183 had no effects on cell adhesion in HeLa cells, it significantly decreased adhesion to laminin, gelatin, and collagen type I in normal human diploid fibroblasts and human trabecular meshwork cells. These effects were also rescued by expression of ITGB1 lacking the 3′-UTR. Targeting of KIF2A by miR-183 resulted in some increase in the formation of cells with monopolar spindles in HeLa cells but not in human diploid fibroblast or human trabecular meshwork cells. The regulation of ITGB1 expression by miR-183 provides a new mechanism for the anti-metastatic role of miR-183 and suggests that this miRNA could influence the development and function in neurosensory organs, and contribute to functional alterations associated with cellular senescence in human diploid fibroblasts and human trabecular meshwork cells.     

Selenoprotein S/SEPS1 Modifies Endoplasmic Reticulum Stress in Z Variant ��1-Antitrypsin Deficiency

Z α₁-antitrypsin (ZAAT) deficiency is a disease associated with emphysematous lung disease and also with liver disease. The liver disease of AAT deficiency is associated with endoplasmic reticulum (ER) stress. SEPS1 is a selenoprotein that, through a chaperone activity, decreases ER stress. To determine the effect of SEPS1 on ER stress in ZAAT deficiency, we measured activity of the grp78 promoter and levels of active ATF6 as markers of the unfolded protein response in HepG2 cells transfected with the mutant form of AAT, a ZAAT transgene. We evaluated levels of NFκB activity as a marker of the ER overload response. To determine the effect of selenium supplementation on the function of SEPS1, we investigated glutathione peroxidase activity, grp78 promoter activity, and NFκB activity in the presence or absence of selenium. SEPS1 reduced levels of active ATF6. Overexpression of SEPS1 also inhibited grp78 promoter and NFκB activity, and this effect was enhanced in the presence of selenium supplementation. This finding demonstrates a role for SEPS1 in ZAAT deficiency and suggests a possible therapeutic potential for selenium supplementation.SEPS1 (selenoprotein S, VIMP, Tanis, or SelS) is a selenoprotein found in the endoplasmic reticulum (ER)³ membrane. SEPS1 participates in the processing and removal of misfolded proteins from the ER to the cytosol, where they are polyubiquitinated and degraded through the proteasome (1). SEPS1 can be induced by ER stress (2) and has been shown in macrophages to be protective from pharmacological ER stress agent-induced apoptosis (3).The endoplasmic reticulum is one of the largest cell organelles. It serves many essential functions, including production of all components of cellular membranes, proteins, lipids, and sterols (4). Only correctly folded proteins are transported out of the ER, whereas incompletely folded proteins are retained in the organelle to complete the folding process or be targeted for destruction. ER stress is defined as an imbalance between the cellular demand for ER function and capacity of the organelle. It is characterized by a number of intracellular responses. These responses include the ER overload response (EOR), the unfolded protein response (UPR), and apoptosis.α₁-Antitrypsin (AAT) deficiency is a disease characterized by early onset emphysema and liver disease (5). The mutant Z form of this autosomal co-dominant disease occurs in >95% of all individuals with AAT deficiency (6). Liver disease occurs in ∼10% of all homozygous neonates who develop hepatitis and cholestasis. A proportion of these children progress to liver failure, requiring liver transplantation (7, 8). Cirrhosis can also occur in adults without a preceding history of childhood liver disease. The mutant Z AAT polymerizes and accumulates in the ER, leaving only 15% of ZAAT secreted (9, 10). This accumulation of abnormal protein in the ER gives rise to ER stress, which is believed to contribute to the liver disease that results from AAT deficiency. The cells respond to this perturbation by inducing the expression of novel genes whose products aim to restore normal ER function (11). SEPS1 is an example of a molecular chaperone that serves to augment the capacity of the ER for protein folding and degradation.In this paper, we investigate the role of SEPS1 in regulating the cellular response to ER stress in HepG2 cells transfected with the ZAAT transgene. We investigate the effect of SEPS1 on the UPR component of this response by measuring grp78 promoter activity, a UPR-up-regulated gene that functions as a molecular chaperone, and by detecting activated ATF6, which occurs downstream to the activation of grp78. The EOR component of ER stress is investigated by measuring NFκB activation.We study the effect of selenium supplementation on the action of SEPS1 to see if the function of this selenoprotein can be enhanced and, if so, through which pathways, looking specifically at grp78 promoter activity, ATF6 activation, and NFκB activation and also at glutathione peroxidase (GPx) activity and at the anti-inflammatory 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) pathway. GPx is a selenoprotein whose activity can be readily assayed. This was used as a measure of selenoprotein activity.The role of SEPS1 in conformational diseases has not been evaluated. These diseases are caused by inherited or acquired modifications in protein structure, where specific proteins undergo a conformational rearrangement, causing deposition within cellular compartments, such as the ER. This can lead to devastating results. AAT deficiency is one such disease, but the group includes Alzheimer, Parkinson, and Huntington diseases and cystic fibrosis.     

Plasminogen Activator Inhibitor-1 Regulates Integrin ��v��3 Expression and Autocrine Transforming Growth Factor �� Signaling

Fibrosis is characterized by elevated transforming growth factor β (TGFβ) signaling, resulting in extracellular matrix accumulation and increased PAI-1 (plasminogen activator inhibitor) expression. PAI-1 induces the internalization of urokinase plasminogen activator/receptor and integrin αvβ3 from the cell surface. Since increased αvβ3 expression correlates with increased TGFβ signaling, we hypothesized that aberrant PAI-1-mediated αvβ3 endocytosis could initiate an autocrine loop of TGFβ activity. We found that in PAI-1 knock-out (KO) mouse embryonic fibroblasts), αvβ3 endocytosis was reduced by ∼75%, leaving αvβ3 in enlarged focal adhesions, similar to wild type cells transfected with PAI-1 small interfering RNA. TGFβ signaling was significantly enhanced in PAI-1 KO cells, as demonstrated by a 3-fold increase in SMAD2/3-containing nuclei and a 2.9-fold increase in TGFβ activity that correlated with an increase in αvβ3 and TGFβ receptor II expression. As expected, PAI-1 KO cells had unregulated plasmin activity, which was only partially responsible for TGFβ activation, as evidenced by a mere 25% reduction in TGFβ activity when plasmin was inhibited. Treatment of cells with an αvβ3-specific cyclic RGD peptide (GpenGRGD) led to a more profound (59%) TGFβ inhibition; a nonspecific RGD peptide (GRGDNP) inhibited TGFβ by only 23%. Human primary fibroblasts were used to confirm that PAI-1 inhibition and β3 overexpression led to an increase in TGFβ activity. Consistent with a fibrotic phenotype, PAI-1 KO cells were constitutively myofibroblasts that had a 1.6-fold increase in collagen deposition over wild type cells. These data suggest that PAI-1-mediated regulation of αvβ3 integrin is critical for the control of TGFβ signaling and the prevention of fibrotic disease.Fibrotic disorders can result from environmental toxins, persistent infection, autoimmune disease, or mechanical injury, leading to the hardening and scarring of tissues. In fibrotic diseases, such as liver cirrhosis, renal fibrosis, and idiopathic lung fibrosis, or in pathological wound healing, such as hypertrophic scarring, scleroderma, and Dupuytren disease, the persistence of myofibroblasts contributes to disease progression by overproduction of extracellular matrix (ECM)² and by excessive contraction (1–3). A shift in the balance of growth factors and cytokines that promote ECM deposition and proteases that degrade matrix often contributes to fibrotic disease (4, 5). Plasmin, a broad spectrum protease that is generated from plasminogen by uPA, is one of the proteases that degrades matrix and activates growth factors and other proteases (6). Since uPA activity is inhibited by PAI-1, the overexpression of PAI-1 results in matrix accumulation. For this reason, PAI-1 is a key prognostic marker for fibrotic disease. PAI-1 exerts its inhibitory activity on uPA by stimulating the endocytosis of the cell surface uPA·uPAR complex through the low density lipoprotein receptor-related protein (7). Integrin αvβ3 is also internalized with the uPA·uPAR·low density lipoprotein receptor-related protein complex (8). After endocytosis, uPAR and integrins are recycled back to the cell surface for another round of binding (8, 9). uPAR and αvβ3 promote cellular attachment and spreading, since they are receptors for the extracellular matrix molecule, vitronectin (10). Thus, cycling of the complex is thought to stimulate the attachment and detachment that is necessary for cell migration (8). Consequently, a shift in the expression of any of these components (PAI-1/uPA/uPAR/αvβ3) can result in either aggressive migration, as seen in cancer invasion, or a persistent increase in cell adhesion and cell tension, as seen in myofibroblasts in fibrotic tissue.The family of TGFβ growth factors has been intensively studied for their role in fibrotic wound healing. Up-regulation of TGFβ results in amplified and persistent overproduction of molecules, such as integrins and PAI-1 and other protease inhibitors (e.g. TIMPs) (2, 3). Up-regulated integrins continue the cycle of TGFβ signaling by participating in the sustained activation of TGFβ from its latent form. To date, studies have found that various αv integrins participate in the activation of TGFβ (αvβ3, αvβ5, αvβ6, and αvβ8), but the mechanism differs (11–15). Integrins can serve as docking proteins to localize proteases that cleave and activate latent TGFβ in the ECM, or they can directly activate latent TGFβ in a protease-independent manner. Recently, it was discovered that latent TGFβ is also activated by mechanical stress generated from an integrin-mediated interaction between myofibroblasts and the ECM, primarily involving αvβ5. The mechanical stress promotes a conformational change that activates the latent TGFβ complex (15). αv integrins also modulate TGFβ signaling through the binding of αvβ3 to TGFβ receptor II (TGFβRII) in the presence of TGFβ. This interaction was shown to promote a dramatic increase in the proliferation of lung fibroblasts and induce invasion of epithelial breast cancer cells (16, 17).Our data establish a role for the PAI-1-mediated control of αvβ3 expression and support a significant role for αvβ3 in TGFβ signaling. Using PAI-1 KO cells, we tested the hypothesis that the absence of PAI-1 would result in the accumulation of αvβ3 on the cell surface, since PAI-1 promotes the endocytosis of uPA·uPAR·αvβ3. PAI-1-mediated endocytosis of β3 was significantly reduced in the PAI-1 KO cells. Correspondingly, we report that β3 accumulated at the cell surface in enlarged β3-containing focal adhesions. Thus, we explored whether the accumulation of αvβ3 on the cell surface had fibrogenic effects even in the absence of profibrotic PAI-1. Our results demonstrate dramatically increased TGFβ activity and an increase in collagen expression in PAI-1 KO cells. Together, these findings suggest that PAI-1 modulates β3 expression and localization and, in turn, TGFβ signaling. Our data reveal that maintaining precise levels of PAI-1 is a key to preventing fibrosis. Understanding the consequence of regulating PAI-1 activity is critical in light of the many clinical therapies currently under development that target PAI-1 (18, 19).     

Membrane Partitioning: ��Classical�� and ��Nonclassical�� Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the "nonclassical" hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (-TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy-enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about -0.5 kcal mol(-1) K(-1). This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔG (hΦ), which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543-552, 2001]. Our results suggest that there is no need for a special effect-the nonclassical hydrophobic effect-to describe partitioning into lipid bilayers.     

Transforming Growth Factor-�� (TGF-��1) Activates TAK1 via TAB1-mediated Autophosphorylation, Independent of TGF-�� Receptor Kinase Activity in Mesangial Cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that signals through the interaction of type I (TβRI) and type II (TβRII) receptors to activate distinct intracellular pathways. TAK1 is a serine/threonine kinase that is rapidly activated by TGF-β1. However, the molecular mechanism of TAK1 activation is incompletely understood. Here, we propose a mechanism whereby TAK1 is activated by TGF-β1 in primary mouse mesangial cells. Under unstimulated conditions, endogenous TAK1 is stably associated with TβRI. TGF-β1 stimulation causes rapid dissociation from the receptor and induces TAK1 phosphorylation. Deletion mutant analysis indicates that the juxtamembrane region including the GS domain of TβRI is crucial for its interaction with TAK1. Both TβRI-mediated TAK1 phosphorylation and TGF-β1-induced TAK1 phosphorylation do not require kinase activity of TβRI. Moreover, TβRI-mediated TAK1 phosphorylation correlates with the degree of its association with TβRI and requires kinase activity of TAK1. TAB1 does not interact with TGF-β receptors, but TAB1 is indispensable for TGF-β1-induced TAK1 activation. We also show that TRAF6 and TAB2 are required for the interaction of TAK1 with TβRI and TGF-β1-induced TAK1 activation in mouse mesangial cells. Taken together, our data indicate that TGF-β1-induced interaction of TβRI and TβRII triggers dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation and not by the receptor kinase activity of TβRI.Members of the transforming growth factor-β (TGF-β)³ superfamily are key regulators of various biological processes such as cellular differentiation, proliferation, apoptosis, and wound healing (1, 2). TGF-β1, the prototype of TGF-β family, is a potent inducer of extracellular matrix synthesis and is well established as a central mediator in the final common pathway of fibrosis associated with progressive kidney diseases (3, 4). Upon ligand stimulation, TGF-β type I (TβRI) and type II (TβRII) receptors form heterotetrameric complexes, by which TβRI is phosphorylated in the GS domain and activated. Smad signaling pathway is well established as a canonical pathway induced by TGF-β1 (5, 6). Receptor-regulated Smads (Smad2 and Smad3) are recruited and activated by the activated TβRI. The phosphorylation in the GS domain (7) and L45 loop (8) of TβRI are crucial for its interaction with receptor-regulated Smads. After phosphorylation, receptor-regulated Smads are rapidly dissociated from TβRI and interact with common Smad (Smad4) followed by nuclear translocation. In addition to the Smad pathway, a recently emerging body of evidence has demonstrated that TGF-β1 also induces various Smad-independent signaling pathways (9–17) by which mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) (18, 19), p38 MAPK (20–22), and extracellular signal-regulated kinase 1/2 (23, 24) can be activated by TGF-β1.TAK1, initially identified as a MAPK kinase kinase 7 (MKKK7 or MAP3K7) in the TGF-β signaling pathway (11, 12), also can be activated by environmental stress (25), proinflammatory cytokines such as IL-1 and TNF-α (26, 27) and lipopolysaccharide (28). For TAK1 activation, phosphorylation at Thr-187 and Ser-192 in the activation loop of TAK1 is essentially required (29–31). TAK1 can transduce signals to several downstream signaling cascades, including the MAPK kinase (MKK) 4/7-JNK cascade, MKK3/6-p38 MAPK cascade, and nuclear factor κB (NF-κB)-inducing kinase-IκB kinase cascade (26–28). A recent report has shown that TAK1 is also activated by agonists of AMP-activated kinase (AMPK) and ischemia, which in turn activates the LKB1/AMPK pathway, a pivotal energy-sensor pathway (32). TAK1 is also involved in Wnt signaling (33). We and others have previously demonstrated that TAK1 is a major mediator of TGF-β1-induced type I collagen and fibronectin expression through activation of the MKK3-p38 MAPK and MKK4-JNK signaling cascades, respectively (34–37). Furthermore, increased expression and activation of TAK1 enhance p38 phosphorylation and promote interstitial fibrosis in the myocardium from 9-day-old TAK1 transgenic mice (37). These data implicate a crucial role of TAK1 in extracellular matrix production and tissue fibrosis. TAK1 is also implicated in regulation of cell cycle (38), cell apoptosis (39–41), and the Smad signaling pathway (42–44). Thus, TAK1 may function as an important regulator and mediator of TGF-β1-induced Smad-dependent and Smad-independent signaling pathways.It has been demonstrated that TAK1 can be activated by the interaction with TAK1-binding protein 1 (TAB1) by in vitro binding assays and in overexpression studies (29–31); however, it is not clear whether TAB1 plays a crucial role in ligand-induced TAK1 activation. In embryonic fibroblasts from TAB1 null mice, IL-1 and TNF-α could induce TAK1-mediated NF-κB and JNK activation (45). TAK1 activation induced by TNF-α, IL-1, and T-cell receptor requires TAB2 or its homologous protein TAB3 (46–50). Although many questions still remain, much progress has been made in understanding the activation mechanism of TAK1 by inflammatory cytokines (46, 47, 51–53). Ligand binding of IL-1 receptor (IL-1R) results in recruitment of MyD88, which serves as an adaptor for IL-1 receptor-associated kinase (IRAK) 1 and 4. Subsequently IRAK1 is hyperphosphorylated and induces interaction with TNF-α receptor-associated factor 6 (TRAF6), resulting in TRAF6 oligomerization. After oligomerization of TRAF6, IRAK1-TRAF6 complex is dissociated from the receptor and associated with TAK1, which is mediated by TAB2 (or TAB3). In this process polyubiquitination of TRAF6 by Ubc13/Uev1A is thought to be critical for the association with TAB2 (or TAB3), which links TAK1 activation (46, 54, 55). In the case of TNF-α stimulation, TNF-α receptors form trimers and recruit adaptor proteins, TRAF2/5, and receptor-interacting protein 1 on the membrane. Ubc13/Uev1A- and TRAF2-dependent polyubiquitination of receptor-interacting protein 1 induce association of TAB2 (or TAB3), which then activates TAK1. Thus, TAB2 is required for ubiquitin-dependent activation of TAK1 by TRAFs. On the other hand, it has been demonstrated that hematopoietic progenitor kinase 1 plays a role as an upstream mediator of TGF-β-induced TAK1 activation, which in turn activates the MKK4-JNK signaling cascade in 293T cells (56, 57). Besides hematopoietic progenitor kinase 1, it has been also suggested that X-linked inhibitor of apoptosis (XIAP) might link TAK1 to TGF-β/BMP receptors through the capability of XIAP to interact with TGF-β/BMP receptors and TAB1 (58). Thus, although various molecules participate in the activation of TAK1, the precise mechanism by which TGF-β1 induces TAK1 activation is incompletely understood. Here, we provide evidence that the association of TAK1 with TGF-β receptors is important for TGF-β1-induced activation of TAK1 in mouse mesangial cells. TGF-β1 stimulation induces interaction of TβRI and TβRII, triggering dissociation of TAK1 from TβRI, and subsequently TAK1 is phosphorylated through TAB1-mediated autophosphorylation, independent of receptor kinase activity of TβRI.